Download CHAPTER 2.1.4 INFECTIOUS HAEMATOPOIETIC NECROSIS

CHAPTER 2.1.4
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INFECTIOUS HAEMATOPOIETIC NECROSIS
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Scope
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Infectious haematopoietic necrosis (IHN) is a viral disease affecting most species of salmonid fish. The principal clinical and economic
consequences of IHN occur at farms rearing fry or juvenile rainbow trout in freshwater where acute outbreaks can result in very high mortality;
however, both Pacific and Atlantic salmon reared in fresh water or sea water can be severely affected. Caused by the rhabdovirus infectious
haematopoietic necrosis virus (IHNV), the disease is typically characterised by gross signs that include lethargy interspersed with bouts of
frenzied, abnormal activity, darkening of the skin, pale gills, ascites, distended abdomen, exophthalmia, and petechial haemorrhages internally and
externally. Internally, fish appear anaemic and lack food in the gut. The liver, kidney and spleen are pale. Histopathological findings reveal
degenerative necrosis in haematopoietic tissues, kidney, spleen, liver, pancreas, and digestive tract. The blood of affected fry shows reduced
haematocrit, leukopenia, degeneration of leukocytes and thrombocytes, and large amounts of cellular debris. For the purpose of this chapter, IHN is
considered to be infection with IHNV.
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For detailed reviews of the disease, see Bootland & Leong (3) or Wolf (27).
Disease information
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2.1. Agent factors
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2.1.1.
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The fish rhabdovirus, IHNV, has a bullet-shaped virion containing a non-segmented, negative-sense, single-stranded RNA genome of
approximately 11,000 nucleotides that encodes six proteins in the following order: a nucleoprotein (N), a phosphoprotein (P), a matrix
protein (M), a glycoprotein (G), a non-virion protein (NV), and a polymerase (L). The presence of the unique NV gene and sequence
similarity with certain other fish rhabdoviruses, such as viral haemorrhagic septicaemia virus, has resulted in creation of the
Novirhabdovirus genus of the family Rhabdoviridae, with IHNV as the type species. The type strain of IHNV is the Western Regional
Aquaculture Center (WRAC) strain available from the American Type Culture Collection (ATCC VR-1392). The GenBank accession
number of the genomic sequence of the WRAC strain is L40883 (26).
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Sequence analysis has been used to compare IHNV isolates from North America, Europe and Asia (6, 8, 12, 14, 18, 22). Within the
historic natural range of the virus in western North America, most isolates of IHNV from Pacific salmon form two genogroups that
are related to geographical location and not to year of isolation or host species. The isolates within these two genogroups show a
relatively low level of nucleotide diversity, suggesting evolutionary stasis or an older host–pathogen relationship. Conversely, isolates
of IHNV from farmed rainbow trout in the USA form a third genogroup with more genetic diversity and an evolutionary pattern
indicative of ongoing adaptation to a new host or rearing conditions. Isolates from farmed rainbow trout in Europe and Asia appear to
have originated from North America, but have diverged somewhat independently.
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On the basis of antigenic studies using neutralising polyclonal rabbit antisera, IHNV isolates form a single serogroup (7). However,
mouse monoclonal antibodies have revealed a number of neutralising epitopes on the glycoprotein (10, 21, 25), as well as the
existence of a non-neutralising group epitope borne by the nucleoprotein (20). Variations in the virulence and host preference of IHNV
strains have been recorded during both natural cases of disease and in experimental infections (9, 15).
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2.1.2.
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IHNV is heat, acid and ether labile. The virus will survive in fresh water for at least 1 month, especially if organic material is present. It
is readily inactivated by common disinfectants and drying (27).
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2.1.3.
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2.1.4.
Aetiological agent, agent strains
Survival outside the host
Stability of the agent (effective inactivation methods)
Life cycle
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Chapter 2.1.4. – Infectious haematopoietic necrosis
Reservoirs of IHNV are clinically infected fish and covert carriers among cultured, feral or wild fish. Virus is shed via faeces, urine,
sexual fluids and external mucus, whereas kidney, spleen, encephalon and the digestive tract are the sites in which virus is most
abundant during the course of overt infection.
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2.2. Host factors
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2.2.1.
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Susceptible species include: rainbow or steelhead trout (Oncorhynchus mykiss), brown trout (Salmo trutta), Pacific salmon including
chinook (O. tshawytscha), sockeye (O. nerka), chum (O. keta), masou (O. masou), and coho (O. kisutch), and Atlantic salmon (Salmo
salar).
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2.2.2.
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Among individuals, there is a high degree of variation in susceptibility to IHNV. The age of the fish is extremely important: the younger
the fish, the more susceptible to disease. As with viral haemorrhagic septicaemia virus, good fish health condition seems to decrease
susceptibility to overt IHN, while co-infections with bacterial diseases (e.g. bacterial coldwater disease), handling and other stressors
can cause subclinical infections to become overt. Fish become increasingly resistant to infection with age until spawning, when they
once again become highly susceptible and may shed large amounts of virus in sexual products. Survivors of IHN demonstrate a strong
protective immunity with the synthesis of circulating antibodies to the virus (17).
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IHN occurs among several species of salmonids with fry being the most highly susceptible stage. Older fish are typically more
resistant to clinical disease. Under natural conditions, most clinical IHN is seen in fry when the water temperature is between 8 and
15°C.
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2.2.5.
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Historically, the geographic range of IHN was limited to western North America, but the disease has spread to Europe and Asia via the
importation of infected fish and eggs. Once IHNV is introduced into a farmed stock, the disease may become established among wild
fish in the watershed.
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Susceptible host species
Susceptible stages of the host
Species or sub-population predilection (probability of detection)
Target organs and infected tissue
Persistent infection with lifelong carriers
Vectors
Known or suspected wild aquatic animal carriers
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2.3. Disease pattern
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Infection with IHNV often leads to mortality due to the impairment of osmotic balance, and occurs within a clinical context of oedema and
haemorrhage. Virus multiplication in endothelial cells of blood capillaries, haematopoietic tissues, and cells of the kidney underlies the
clinical signs. High levels of virus are shed from infected juvenile fish.
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2.3.1.
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The transmission of IHNV between fish is primarily horizontal; however, cases of vertical or ‘egg-associated’ transmission have been
recorded. Horizontal transmission is typically by direct exposure, but invertebrate vectors have been proposed to play a role in some
cases. Although egg-associated transmission is significantly reduced by the now common practice of surface disinfection of eggs
with an iodophor solution, it is, however, the only mechanism accounting for the appearance of IHN in new geographical locations
among alevins originating from eggs that were incubated and hatched in virus-free water.
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Transmission mechanisms
Prevalence
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Chapter 2.1.4. – Infectious haematopoietic necrosis
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Geographical distribution
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Depending on the species of fish, rearing conditions, temperature, and, to some extent, the virus strain, outbreaks of IHN may range
from explosive to chronic. Losses in acute outbreaks will exceed several per cent of the population per day and cumulative mortality
may reach 90–95% or more. In chronic cases, losses are protracted and fish in various stages of disease can be observed in the
pond. The disease is made more severe by co-infection with certain bacterial pathogens, such as Flavobacterium psychrophilum.
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The most important environmental factor affecting the progress of IHN is water temperature. Clinical disease typically occurs
between 8°C and 15°C under natural conditions.
Mortality and morbidity
Environmental factors
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2.4. Control and prevention
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Control methods for IHN currently rely on avoidance of exposure to the virus through the implementation of strict control policies and sound
hygiene practices (23). The thorough disinfection of fertilised eggs, the use of virus-free water supplies for incubation and rearing, and the
operation of facilities under established biosecurity measures are all critical for preventing IHN at a fish production site.
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Vaccination
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At present, vaccination of salmonids against IHN is at an early stage of development; however, a range of vaccine preparations have
shown promise in both laboratory and field trials (13, 23, 24). Both autogenous, killed vaccines and a novel DNA vaccine have been
approved for commercial use in Atlantic salmon net-pen aquaculture on the west coast of North America where a vaccine can be
delivered economically by injection. However, the application of the available IHN vaccines to millions of small fish will require
additional research on novel mass delivery methods.
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Chemotherapy
Immunostimulation
Resistance breeding
Restocking with resistant species
Blocking agents
Disinfection of eggs and larvae
General husbandry practices
Sampling
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3.1. Selection of individual specimens
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3.2. Preservation of samples for submission
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3.3. Pooling of samples
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3.4. Best organs or tissues
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3.5. Comment on fish/tissues that are not appropriate (i.e. when it is never possible to detect)
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Diagnostic methods
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The ‘Gold Standard’ for detection of IHNV is the isolation of the virus in cell culture followed by its immunological or molecular identification. While
the other diagnostic methods listed below can be used for confirmation of the identity of virus isolated in cell culture or for confirmation of overt
infections in fish, they are not approved for use as primary surveillance methods for obtaining or maintaining approved IHN-free status.
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Due to substantial variation in the strength and duration of the serological responses of fish to virus infections, the detection of fish antibodies to
viruses has not thus far been accepted as a routine diagnostic method for assessing the viral status of fish populations. In the future, validation of
serological techniques for diagnosis of fish virus infections could render the use of fish serology more widely acceptable for diagnostic purposes.
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4.1. Field diagnostic methods
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During outbreaks, fish are typically lethargic with bouts of frenzied, abnormal activity such as spiral swimming and flashing. Affected fish
exhibit darkening of the skin, pale gills, ascites, distended abdomen, exophthalmia, and petechial haemorrhages internally and externally. A
trailing faecal cast is observed in some species. Spinal deformities are present among some of the surviving fish.
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Clinical signs
Behavioural changes
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4.2. Clinical methods
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Internally, fish appear anaemic and lack food in the gut. Ascitic fluid and petechiae are observed in the organs of the body cavity. The liver,
kidney and spleen are pale. Histopathological findings reveal degenerative necrosis in haematopoietic tissues, kidney, spleen, liver, pancreas
and digestive tract. The blood of affected fry shows reduced haematocrit, leukopenia, degeneration of leukocytes and thrombocytes, and
large amounts of cellular debris. The cellular debris, termed necrobiotic bodies, can be seen in stained tissue imprints from the anterior
kidney and has diagnostic value. As with other haemorrhagic viraemias of fish, blood chemistry is altered in severe cases.
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Gross pathology
Clinical chemistry
Microscopic pathology
Wet mounts
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Chapter 2.1.4. – Infectious haematopoietic necrosis
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4.2.5.
Smears
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Electron microscopy of virus-infected cells reveals bullet-shaped virions of approximately 150–190 nm in length and 65–75 nm in
width (27). The virions are visible at the cell surface or within vacuoles or intracellular spaces after budding through cellular
membranes. The virion possesses an outer envelope containing host lipids and the viral glycoprotein spikes that react with
immunogold staining to decorate the virion surface.
Fixed sections
Electron microscopy/cytopathology
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4.3. Agent detection and identification methods
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The traditional procedure for detection of IHNV is based on virus isolation in cell culture. Confirmatory identification may be achieved by use
of immunological (neutralisation, indirect fluorescent antibody test or enzyme-linked immunosorbent assay), or molecular (DNA probe or
polymerase chain reaction) methods (1, 2, 4, 5, 11, 16, 19, 26)
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4.3.1.1.1. Wet mounts
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4.3.1.1.3. Fixed sections
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4.3.1.2.
Agent isolation and identification
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4.3.1.2.1. Isolation of IHNV in cell culture
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Cell lines to be used: EPC and BF-2
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Inoculation of cell monolayers
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i)
Following the virus extraction procedure described in Section B.3.2 of Chapter 2.1, make an additional tenfold dilution of
the 1/10 organ homogenate supernatants and transfer an appropriate volume of each of the two dilutions to 24-hour-old
cell monolayers. Inoculate at least 2 cm2 of drained cell monolayer with 100 µl of each dilution.
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Allow to adsorb for 0.5–1 hour and, without withdrawing the inoculum, add the cell culture medium buffered at pH 7.6 and
supplemented with 2% fetal calf serum (FCS) (1 ml/well for 24-well cell culture plates), and incubate at 15°C.
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If required, the inoculum may be pre-incubated with neutralising antiserum against infectious pancreatic necrosis virus
(IPNV) or other endemic viruses, as previously described.
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Monitoring incubation
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Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination at
×40–100 magnification for 7 days. The use of a phase-contrast microscope is recommended.
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Maintain the pH of the cell culture medium between 7.3 and 7.6 during incubation. This can be achieved, especially in open
plates, by using cell culture medium containing sodium bicarbonate that is further buffered by addition of Tris or HEPES
buffer (N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid).
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If a cytopathic effect (CPE) appears in cell cultures inoculated with dilutions of the fluids or homogenates, identification
procedures must be undertaken immediately (see below).
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If no CPE develops in the inoculated cultures (despite normal progression of CPE in the virus controls), the inoculated
cultures should be subcultured for a further 7 days. Should the virus control fail to develop CPE, the process should be
repeated with fresh cell cultures and new batches of samples.
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Subcultivation procedures
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Collect aliquots of cell culture medium from all monolayers inoculated with various dilutions of fluid or organ homogenate
samples.
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If required, repeat neutralisation of IPNV or other endemic viruses as previously described.
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Inoculate cell monolayers as described above.
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Incubate and monitor as described above.
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If no CPE occurs, the test may be declared negative.
4.3.1.2.2. Identification of IHNV isolated in cell culture
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Neutralisation test
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Collect the medium from cultures exhibiting CPE that involves at least 25% of the cell monolayer and centrifuge it at
2000 g for 15 minutes at 4°C to remove cell debris.
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In parallel, other neutralisation tests must be performed against:
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a known isolate of IHNV (positive neutralisation test).
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a heterologous virus (negative neutralisation test).
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The neutralisation test procedure
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Dilute the virus-containing medium from 10–2 to 10–4.
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Mix aliquots (for example 200 µl) of each virus dilution with equal volumes of an appropriate dilution of rabbit polyclonal or
mouse monoclonal antibody (MAb) against IHNV, and similarly treat aliquots of each virus dilution with cell culture medium.
(The neutralising antibody solution must have a 50% plaque reduction titre of at least 2000).
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Incubate all the mixtures at 15°C for 1 hour.
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Transfer aliquots of each of the above mixtures on to drained cell monolayers (inoculate two cell cultures per dilution) and
allow adsorption to occur for 0.5–1 hour at 15°C; 24- or 12-well cell culture plates are suitable for this purpose, using a
50 µl inoculum.
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When adsorption is complete, add cell culture medium, supplemented with 2% FCS and buffered at pH 7.4–7.6, to each well
and incubate at 15°C.
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Check the cell cultures for the onset of CPE and read the result as soon as it occurs in non-neutralised controls (cell
monolayers being protected in positive neutralisation controls). Results are recorded either after a simple microscopic
examination (phase-contrast preferable) or after discarding the cell culture medium and staining the cell monolayers with
a solution of 1% crystal violet in 10% buffered formalin.
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The tested virus is identified as IHNV when CPE is prevented or noticeably delayed in the cell cultures that received the
virus suspension treated with the IHNV-specific antibody, whereas CPE is present in the untreated sample.
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In the absence of significant neutralisation when IHNV is suspected, it is advisable to conduct an indirect fluorescent antibody
test (IFAT) as antigenic drift has been observed in the neutralising epitopes on the IHNV glycoprotein, resulting in occasional
failures of the neutralisation test for certain strains of the virus.
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If required, a similar neutralisation test may be performed using antibodies to IPNV or other enzootic viruses to ensure
that no contaminant has escaped the first assay.
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4.3.1.2.3. Antibody-based antigen detection methods
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4.3.1.2.3.1 Indirect fluorescent antibody test
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Preparation of monolayers
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i)
Prepare monolayers of cells in 2 cm2 wells of plastic cell culture plates or on glass cover-slips to reach around 80%
confluency within 24 hours of incubation at 22°C. Seed six cell monolayers per virus isolate to be tested, plus two for
positive and two for negative controls. The FCS content of the cell culture medium can be reduced to 2–4%.
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When the cell monolayers are ready for infection (i.e. on the same day or on the day after seeding), inoculate the virus
suspensions to be identified by making tenfold dilution steps directly in the cell culture wells or flasks.
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Dilute the control virus suspension of IHNV in a similar way, in order to obtain a virus titre of about 5000–
10,000 infectious units/ml in the cell culture medium.
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Incubate at 15°C for 24 hours.
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Remove the cell culture medium, rinse once with 0.01 M phosphate buffered saline (PBS), pH 7.2, then three times briefly
with cold acetone (stored at –20°C) for glass cover-slips or a mixture of 30% acetone and 70% ethanol, also at –20°C,
for plastic wells.
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Let the fixative act for 15 minutes. A volume of 0.5 ml is adequate for 2 cm2 of cell monolayer.
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Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or freeze at –20°C.
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The indirect fluorescent antibody procedure
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Prepare a solution of antibody against IHNV in PBS containing 0.05% Tween 80 (PBST) at the appropriate dilution
(established previously or given by the reagent supplier). The antibody used should be able to bind to all isolates of the
virus.
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Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and remove this buffer completely after
the last rinsing.
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Treat the cell monolayers with the antibody solution for 1 hour at 37°C in a humid chamber and do not allow evaporation to
occur. The volume of solution to be used is 0.25 ml/2 cm2 well.)
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Rinse four times with PBST as above.
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Treat the cell monolayers for 1 hour at 37°C with a solution of fluorescein isothiocyanate (FITC)-conjugated antibody to the
immunoglobulin used in the first layer and prepared according to the instructions of the supplier. These FITC antibodies
are most often rabbit or goat antibodies.
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Rinse four times with PBST.
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Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using glycerol saline at pH 8.5
prior to microscopic observation.
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viii) Examine under incident UV light using a microscope with a ×20–40 objective lens having a high numerical aperture.
Positive and negative controls must be found to give the expected results prior to any other observation.
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4.3.1.2.3.2 Enzyme-linked immunosorbent assay
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Preparation of microplates
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Coat the wells of microplates designed for enzyme-linked immunosorbent assays (ELISAs) with appropriate dilutions of
immunoglobulins (Ig) specific for IHNV, in 0.01 M PBS, pH 7.2 (200 µl/well). The Ig may be polyclonal or monoclonal, most
often from rabbit or mouse. For the identification of IHNV, MAbs specific for certain domains of the nucleocapsid (N)
protein are suitable.
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Incubate overnight at 4°C.
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Rinse four times with 0.01 M PBS containing 0.05% Tween 20 (PBST).
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Block with skim milk (5% in PBST) or other blocking solution for 1 hour at 37°C (200 µl/well).
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Rinse four times with PBST.
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The enzyme-linked immunosorbent assay procedure
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i)
Add 2% Triton X-100 to the virus suspension to be identified.
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Dispense 100 µl/well of two- or four-step dilutions of the virus to be identified and of a known IHNV control, and allow to
react with the coated antibody to IHNV for 1 hour at 20°C.
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Rinse four times with PBST.
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Add to the wells either biotinylated polyclonal rabbit antiserum to IHNV or biotinylated mouse MAb to an N protein epitope
different from the one recognised by the coating MAb.
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Incubate for 1 hour at 37°C.
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Rinse four times with PBST.
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Add streptavidin-conjugated horseradish peroxidase to those wells that have received the biotin-conjugated antibody, and
incubate for 1 hour at 20°C.
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viii) Rinse four times with PBST.
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Add the substrate and chromogen. Stop the course of the test when positive controls react, and read the results.
4.3.1.2.4. Molecular techniques
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4.3.1.2.4.1 DNA probe
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Infect cells with virus isolates
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Trypsinise EPC (or other) cells and resuspend to 1.0 × 106 cells per ml with MEM-5-T (5% FBS [foetal bovine serum], Tris
buffer). Transfer 1 ml of cell suspension to each well of a 24-well tissue culture plate and incubate the plate at an
appropriate temperature.
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On the day after seeding cells, remove the fluid from each cell monolayer. Infect cells with positive control (IHNV) and with
the unknown fish virus isolates in 1 ml total volume to provide a multiplicity of infection (m.o.i.) of 10 PFU per cell. Negative
control cells receive 1 ml MEM-5-T. Incubate for 2 hours at 15–20°C on a rocker platform.
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iii)
Incubate infected and control cells at 15°C overnight. The desired product is the N gene mRNA, and maximum production
occurs at about 12 hours post-infection at high m.o.i. Thus, mRNA production is optimum when infected cells are at an early
stage of virus replication, and RNA can be extracted from cells at the first sign of any CPE. Alternatively, use recently
infected cells from the virus isolation procedure.
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Make or purchase the following solutions:
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Prehybridisation buffer
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Deionised water (DEPC-treated; autoclaved)
10 × Denhardt’s solution
2 × SSC (standard saline citrate)
1% SDS (sodium dodecyl sulfate)
0.1 mg/ml SSS (sonicated salmon sperm) DNA
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Hybridisation solution
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Prehybridisation buffer
Biotinylated DNA probe
(Store at –20°C; may re-use up to five times)
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69.5 ml
10 ml of 100 × stock
10 ml of 20 × stock
10 ml of 10% stock
0.5 ml stock
10 ml
100 ng/ml
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Post-hybridisation solution
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2 × SSC
0.1% SDS
Deionised water (DEPC-treated; autoclaved)
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100 × Denhardt’s solution
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Bovine serum albumin
Polyvinylpyrrolidone 360
Ficoll 400
Deionised water (DEPC-treated; autoclaved)
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Sonicated salmon sperm DNA (SSS DNA at 20 mg/ml)
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Transfer 0.5 ml of SSS DNA (20 mg/ml) into vials and place in boiling water for 10 minutes. Cool vials in crushed ice and store at –
20°C. When needed, add 0.5 ml to prehybridisation buffer.
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20 × standard saline citrate (20 × SSC)
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NaCl
Citric acid
Deionised water (DEPC-treated; autoclaved)
(Adjust to pH 7.0 with HCl, autoclaved)
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10 × standard saline citrate (10 × SSC)
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NaCl
43.82 g
Citric acid
22.05 g
Deionised water (DEPC-treated; autoclaved)
up to 500 ml
(Adjust to pH 7.0 with HCl, autoclaved OR dilute 1/2 from 20 × SSC using deionised water, autoclaved)
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10% sodium dodecyl sulphate (10% SDS)
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Lauryl sulphate sodium salt
Deionised water (DEPC-treated; autoclaved)
(Adjust to pH 7.2. Do not autoclave this solution!)
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Streptavidin/alkaline phosphate conjugate (SA/AP)
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0.1 µg/ml streptavidin/alkaline phosphatase conjugate. Prepare by diluting SA/AP 1/1000 in buffer A. (May re-use this solution up
to five times, store at 4°C.)
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Buffer A
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0.1 M Tris, pH 7.5
0.1 M NaCl
2 mM MgCl2
0.05% Triton X-100
Deionised water (DEPC-treated; autoclaved)
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Buffer B
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0.1 M Tris, pH 9.5
0.1 M NaCl
50 mM MgCl2
Deionised water (DEPC-treated; autoclaved)
Manual of Diagnostic Tests for Aquatic Animals 2009
50 ml of 20 × stock
5 ml of 10% stock
up to 500 ml
50 g
50 g
50 g
up to 500 ml
87.65 g
44.11 g
up to 500 ml
10.0 g
up to 100 ml
50 ml of 1 M stock
10 ml of 5 M stock
1 ml of 1 M stock
0.25 ml
up to 500 ml
50 ml of 1 M stock
10 ml of 5 M stock
25 ml of 1 M stock
up to 500 ml
9
Chapter 2.1.4. – Infectious haematopoietic necrosis
359
5 M NaCl
360
361
362
NaCl
Deionised water (DEPC-treated; autoclaved)
(autoclave this solution)
363
1 M Tris buffer, pH 9.5
364
365
366
367
Tris base
Tris HCl
Deionised water (DEPC-treated; autoclaved)
(Adjust to pH 9.5, then autoclave)
368
1 M Tris buffer, pH 7.5
369
370
371
372
Tris base
Tris HCl
Deionised water (DEPC-treated; autoclaved)
(Adjust to pH 7.5, then autoclave)
373
Chloroform
374
Chloroform (Store at –20°C until needed)
375
Isopropyl alcohol
376
2-propanol (isopropyl alcohol). Use undiluted for precipitation of RNA.
377
Trizol
378
379
RNA isolation reagent, store at 2–8°C in the dark. Invitrogen Catalog No. 15596-026. Caution: Contains guanidinium
isothiocyanate and phenol.
380
Alkaline phosphatase conjugate substrate kit
381
(NOTE: This product contains dimethylformamide. Use in area with good ventilation.)
382
383
384
Dissolve alkaline phosphatase (AP) colour development buffer in 1 litre volume of distilled deionised water. Filter-sterilise then
store at 4°C. Immediately before use, add 0.4 ml of AP colour reagent A and 0.4 ml AP colour reagent B to 39.2 ml colour
development buffer at room temperature.
385
Prepare biotinylated oligonucleotide probe
386
387
388
389
390
391
i)
392
Extraction of mRNA from infected cells
393
394
i)
Always wear protective gloves to avoid contaminating solutions with RNase from skin and to avoid injury. Provide adequate
ventilation, especially for RNA extraction steps with Trizol and for dimethylformamide in colour development solutions.
395
396
397
ii)
Remove culture medium from infected cells and add 1.0 ml Trizol to each well. Replace lid and place plates on a rocker
platform for 5–10 minutes at room temperature to digest the cells. During incubation, load 100 µl chloroform into
siliconised 1.7 ml centrifuge tubes, keep on ice.
398
399
iii)
Triturate cell debris by pipetting with a sterile 1 ml pipette (5×), then transfer solution from each well into a separate tube.
Vortex the tubes (about 3 seconds each), store on crushed ice for 5 minutes to allow phase separation.
400
401
iv)
Centrifuge the suspension at 12,500 g for 15 minutes at 4°C. The RNA will remain in the clear aqueous phase while DNA and
protein will be left in the lower red phenol phase.
10
146.1 g
up to 500 ml
54.7 g
7.6 g
up to 500 ml
11.8 g
63.5 g
up to 500 ml
The biotinylated probe (5’-CTT-GTT-TTG-GCA-GTA-TGT-GGC-CAT-CTT-GTC-3’) is made using the 30-nucleotide sequence
identified by Deering et al. (4). However, three nucleotides containing biotin can be conveniently added to the 5’ end during
DNA synthesis rather than by a subsequent terminal transferase reaction at the 3’ end. This antisense probe is
complementary to a conserved region in the middle of the IHNV nucleoprotein gene mRNA. It should react only with IHNV
and should recognise all isolates of the virus. The final concentration of the biotinylated probe will be 0.1 µg/ml in
hybridisation solution.
Manual of Diagnostic Tests for Aquatic Animals 2009
Chapter 2.1.4. – Infectious haematopoietic necrosis
402
v)
During step iv, load another set of tubes with 0.5 ml of absolute isopropyl alcohol, store on ice.
403
404
405
vi)
Carefully transfer the upper aqueous phase, which contains the RNA (about 0.5 ml), to a tube that contains an equal
volume (0.5 ml) of absolute isopropyl alcohol. Vortex tubes for 1 second, and chill tubes on ice for 15 minutes to precipitate
RNA.
406
407
vii)
Centrifuge the mixture at 12,500 g for 15 minutes at 4°C. Remove as much fluid from the pellet as possible. Partially dry the
pellet following the manufacturer’s instructions.
408
409
viii) Prepare a nitrocellulose membrane (0.45 µm pore size): wet in distilled deionised water for 1 minute, pour water off, then
soak for at least 5 minutes in 10 × SSC.
410
ix)
Warm the prehybridisation buffer to 55°C in a water bath.
411
412
x)
Add 170 µl distilled deionised, ribonuclease-free (DEPC-treated and autoclaved) or molecular-biology grade water to the
RNA pellets. Mix the contents and warm the tubes in a heat block at 65°C for 15–20 minutes (RNA pellets should dissolve).
413
414
xi)
Install the membrane in 96-well vacuum blotting device, attach vacuum pump hoses and add 200 µl of 10 × SSC to each
well to insure that the membrane is not dry when the RNA is added.
415
416
xii)
Heat IHNV PCR products (if used as positive control instead of RNA from IHNV-infected cells) to 95°C for 5 minutes to
denature the double-stranded DNA, and then place tubes directly on ice.
417
418
xiii) Add 170 µl of 20 × SSC into microcentrifuge tubes containing the dissolved pellets of RNA in 170 µl of water (tubes now
contain 340 µl of 10 × SSC). Store on ice.
419
420
421
xiv) Add 100 µl of each RNA solution to wells of a blotting device that contain 200 µl of 10 × SSC. Blot positive controls last.
Apply vacuum for about 1 minute until fluid is pulled through the membrane. Remove membrane and transfer to thick filter
paper wetted with 10 × SSC.
422
xv)
Cut the membrane into sections if required and label the membranes in one corner.
423
424
425
xvi)
Place the membrane(s) between dry sheets of blotting paper and microwave for 60 seconds on high power to attach
nucleic acids to the membrane. Include a beaker of water in the microwave. (Ultraviolet radiation or other suitable
methods may be used.)
426
Hybridisation of probe to RNA on nitrocellulose membrane
427
428
i)
Place the membranes (spots up) in a hybridisation pouch, bottle or plastic bag. Add 10 ml prehybridisation buffer to each
membrane. Prehybridise for 1 hour at 55°C in a shaker water bath.
429
430
ii)
Thaw the probe solution and add 100 µl to prehybridisation buffer. React the membranes in probe solution for 1 hour in
shaker water bath at 55°C.
431
iii)
Remove probe solutions and store in tubes at –20°C for re-use up to five times.
432
433
434
435
iv)
Rinse the membranes with 40 ml of post-hybridisation solution. Discard the solution then add a fresh 40 ml of posthybridisation solution. Wash for 15 minutes on a rocker platform at room temperature. Wash two more times (40 ml) for
15 minutes each at room temperature on a rocker platform. Put the container with the membranes into a 55°C water bath
with pre-warmed post-hybridisation solution (55°C) for 15 minutes.
436
v)
Rinse the membranes briefly with 40 ml of buffer A.
437
Colour development of biotinylated probe
438
439
i)
Incubate the membranes in a solution containing 40 µl streptavidin/alkaline phosphatase in 40 ml buffer A for 30 minutes
at room temperature on a rocker platform. This solution can be re-used at least five times.
440
441
ii)
Rinse the membranes briefly (40 ml) then wash twice using 40 ml buffer A incubated for 7 minutes at room temperature on
a rocker platform.
442
iii)
Wash twice in 40 ml buffer B incubated for 7 minutes at room temperature on a rocker platform.
443
444
445
446
iv)
Warm the colour development buffer prepared according to the manufacturer’s protocol. Buffer should be filter-sterilised
through a 0.2 µm membrane and stored at 4°C. Immediately before use, add 0.4 ml alkaline phosphatase (AP) colour
reagent A and 0.4 ml AP colour reagent B to 39.2 ml AP colour development buffer at room temperature. Add 40 ml colour
development solution containing nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to each
Manual of Diagnostic Tests for Aquatic Animals 2009
11
Chapter 2.1.4. – Infectious haematopoietic necrosis
container with the membranes. Incubate at room temperature on a rocker platform and observe for colour development
(up to 15 minutes).
447
448
449
450
v)
Wash the membranes in distilled deionised water for 10 minutes, and change the water at least once to remove excess
colour development solution. Store membranes in distilled deionised water until photographs can be taken (if desired).
451
4.3.1.2.4.2 Polymerase chain reaction
452
Viral RNA preparation
453
454
455
456
457
i)
458
Reverse-transcription and first round PCR protocol
459
i)
Prepare a master mix for the number of samples to be analysed. Work under a hood and wear gloves.
460
461
462
463
ii)
The master mix for one 50 µl reverse-transcription PCR is prepared as follows: 23.75 µl ribonuclease-free (DEPC-treated)
or molecular-biology grade water; 5 µl 10 × buffer; 5 µl 25 mM MgCl2; 5 µl 2 mM dNTP; 2.5 µl (20 pmoles/µl) Upstream Primer
5’-AGA-GAT-CCC-TAC-ACC-AGA-GAC-3’; 2.5 µl (20 pmoles/µl) Downstream Primer 5’-GGT-GGT-GTT-GTT-TCC-GTG-CAA-3’;
0.5 µl Taq polymerase (5 U/µl); 0.5 µl AMV reverse transcriptase (9 U/µl); 0.25 µl RNasin (39 U/µl).
464
iii)
Centrifuge the tubes briefly (10 seconds) to make sure the contents are at the bottom.
465
466
467
iv)
Place the tubes in the thermal cycler and start the following cycles – 1 cycle: 50°C for 30 minutes; 1 cycle: 95°C for
2 minutes; 30 cycles: 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 60 seconds; 1 cycle: 72°C for 7 minutes and soak
at 4°C.
468
469
vi)
Visualise the 693 bp PCR amplicon by electrophoresis of the product in 1.5% agarose gel with ethidium bromide and
observe using UV transillumination.
470
471
472
473
474
475
NOTE: These PCR primers have been modified from previous editions of this Aquatic Manual to target the central portion of the
IHNV G gene (6), although other primer sets can be used for amplification of portions of the N or G genes of IHNV (26). However,
the primer sequences used here have been shown to be conserved among all known isolates of IHNV and are not present in the G
gene of the related fish rhabdoviruses, viral haemorrhagic septicaemia virus or hirame rhabdovirus. Additionally, the new
primers produce an amplicon that can be used as a template for sequence analysis of the ‘mid-G’ region of the IHNV genome for
epidemiological purposes (6, 14).
Total RNA from infected cells is extracted using a phase-separation method (e.g. phenol-chloroform or TRIZOL, Invitrogen)
or by RNA affinity spin columns (e.g. RNeasy Total RNA kit, Qiagen) used according to the manufacturer’s instructions.
While all of these methods work well for drained cell monolayers or cell pellets, RNA binding to affinity columns can be
affected by salts present in tissue culture media and phase-separations methods should be used for extraction of RNA
from cell culture fluids.
4.3.1.2.5. Diagnostic methods for IHN in fish infected with IHNV
476
Virus isolation with subsequent identification
477
478
Sampling procedures
479
See the following sections in Chapter 2.1:
480
B.1 for the selection of fish specimens
481
B.2 for the selection of materials sampled.
482
Processing of organ samples
483
See the following sections in Chapter 2.1:
484
B.3.1 for transportation
485
B.3.2 for virus extraction and obtaining organ homogenates.
486
Virus isolation and identification
487
As outlined above
Indirect fluorescent antibody test
488
Processing of organ samples
489
12
Manual of Diagnostic Tests for Aquatic Animals 2009
Chapter 2.1.4. – Infectious haematopoietic necrosis
490
i)
Bleed the fish thoroughly.
491
ii)
Make kidney imprints on cleaned glass slides or at the bottom of the wells of a plastic cell culture plate.
492
493
iii)
Store the kidney pieces (as indicated in Chapter 2.1 Section B.3.1) together with the other organs required for virus
isolation in case this becomes necessary later.
494
iv)
Allow the imprint to air-dry for 20 minutes.
495
Indirect fluorescent antibody procedure
496
i)
Fix with acetone or ethanol/acetone and dry as indicated in Section 4.3.1.2.3 Indirect fluorescent antibody.
497
498
ii)
Rehydrate the preparations and block with 5% skim milk or 1% bovine serum albumin, in phosphate buffered saline
containing 0.05% Tween 80 (PBST) for 30 minutes at 37°C.
499
iii)
Rinse four times with PBST.
500
501
iv)
Treat the imprints with the solution of antibody to IHNV and rinse as indicated in 4.3.1.2.3 Indirect fluorescent
antibody.
502
v)
Block and rinse as described previously.
503
vi)
Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as indicated above.
504
If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described above.
505
Enzyme-linked immunosorbent assay
506
Sampling procedures
507
See the following sections in Chapter 2.1:
508
B.1 for the selection of fish specimens
509
B.2 for the selection of materials sampled.
510
Processing of organ samples
511
See the following sections in Chapter 2.1:
512
B.3.1 for transportation
513
B.3.2 for virus extraction and obtaining organ homogenates.
514
Microplate processing
515
As described in Section 4.3.1.2.3 Enzyme-linked immunosorbent assay.
516
The enzyme-linked immunosorbent assay procedure
517
i)
Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
518
519
ii)
Treat the remaining part of the homogenate with 2% Triton X-100 as above and 2 mM of phenyl methyl sulfonide
fluoride; mix gently.
520
iii)
Complete the other steps of the procedure described in 4.3.1.2.3 Enzyme-linked immunosorbent assay.
521
522
If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described in Section
4.3.1.2.1.
523
Polymerase chain reaction
524
Sampling procedures
525
See the following sections in Chapter 2.1:
526
B.1 for the selection of fish specimens
527
B.2 for the selection of materials sampled.
528
Processing of organ samples
529
See the following sections in Chapter 2.1:
Manual of Diagnostic Tests for Aquatic Animals 2009
13
Chapter 2.1.4. – Infectious haematopoietic necrosis
530
B.3.1 for transportation
531
B.3.2 for virus extraction and obtaining organ homogenates.
532
Viral RNA preparation
533
i)
Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
534
535
ii)
Extract RNA from the tissue homogenate using methods described above or by use of other commercially available
kits (19).
536
The polymerase chain reaction procedure
537
Perform the remaining steps of the PCR procedure described above.
538
If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described above.
4.3.1.2.4. Agent purification
539
–
540
541
4.3.2.
542
–
14
Serological methods
Manual of Diagnostic Tests for Aquatic Animals 2009
Chapter 2.1.4. – Infectious haematopoietic necrosis
543
5.
Rating of tests against purpose of use
544
545
546
547
548
549
550
The methods currently available for surveillance, detection, and diagnosis of IHNV are listed in Table 5.1. The designations used in the Table indicate:
a = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity; b = the method is a
standard method with good diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or other
factors severely limits its application; and d = the method is presently not recommended for this purpose. These are somewhat subjective as
suitability involves issues of reliability, sensitivity, specificity and utility. Although not all of the tests listed as category a or b have undergone
formal standardisation and validation (see Chapter 1.1.2), their routine nature and the fact that they have been used widely without dubious results,
makes them acceptable.
551
Table 5.1. Methods for targeted surveillance and diagnosis
Method
Targeted surveillance
Presumptive
diagnosis
Confirmatory
diagnosis
Gametes
Fry
Juveniles
Adults
Gross signs
d
c
c
d
b
d
Virus isolation in cells with
confirmatory ID
a
a
a
a
a
a
Direct LM on tissue imprints
d
c
d
d
b
c
Histopathology of tissues and
organs
d
c
d
d
b
c
Transmission EM of tissues
d
d
d
d
b
c
Antibody-based assays to
detect IHNV antigens
d
c
c
c
a
b
DNA probes to detect IHNV
nucleic acids
d
c
c
c
a
a
PCR of tissue extracts
b
b
b
b
a
a
Direct sequencing of nucleic
acids from tissues
d
d
d
d
c
a
LM = light microscopy; EM = electron microscopy; PCR = polymerase chain reaction.
552
553
6.
Test(s) recommended for targeted surveillance to declare freedom from infectious haematopoietic necrosis
554
555
556
The method for targeted surveillance to declare freedom from IHN is isolation of virus in cell culture. For this purpose, the most susceptible stages
of the most susceptible species should be examined. Reproductive fluids and tissues collected from adult fish of a susceptible species at spawning
should be included in at least one of the sampling periods each year.
Manual of Diagnostic Tests for Aquatic Animals 2009
15
Chapter 2.1.4. – Infectious haematopoietic necrosis
557
7.
Corroborative diagnostic criteria
558
7.1. Definition of suspect case
559
560
561
A suspect case is defined as the presence of typical, gross clinical signs of the disease in a population of susceptible fish, OR a typical
internal histopathological presentation among susceptible species, OR typical cytopathic effect in cell culture without identification of the
agent, OR a single positive result from one of the diagnostic assays ranked as ‘a’ or ‘b’ in Table 1.
562
7.2. Definition of confirmed case
563
564
565
A confirmed case is defined as a suspect case that has EITHER: 1) produced typical cytopathic effect in cell culture with subsequent
identification of the agent by one of the antibody-based or molecular tests listed in Table 1, OR: 2) a second positive result from a different
diagnostic assay ranked as ‘a’ or ‘b’ in the last column of Table 1
566
8.
References
567
568
1.
ARAKAWA C.K., DEERING R.E., HIGMAN K.H., OSHIMA K.H., O’HARA P.J. & WINTON J.R. (1990). Polymerase chain reaction (PCR) amplification of a
nucleoprotein gene sequence of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 8, 165–170.
569
570
2.
ARNZEN J.M., RISTOW S.S., HESSON C.P. & LIENTZ J. (1991). Rapid fluorescent antibody tests for infectious hematopoietic necrosis virus (IHNV)
utilizing monoclonal antibodies to the nucleoprotein and glycoprotein. J. Aquat. Anim. Health, 3, 109–113.
571
572
3.
BOOTLAND L.M. & LEONG J.C. (1999). Infectious hematopoietic necrosis virus. In: Fish Diseases and Disorders, Volume 3: Viral, Bacterial and
Fungal Infections, Woo P.T.K. & Bruno D.W., eds. CAB International, Oxon, UK, 57–121.
573
574
4.
DEERING R.E., ARAKAWA C.K., OSHIMA K.H., O’HARA P.J., LANDOLT M.L. & WINTON J.R. (1991). Development of a biotinylated DNA probe for detection and
identification of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 11, 57–65.
575
5.
DIXON P.F. & HILL B.J. (1984). Rapid detection of fish rhabdoviruses by the enzyme-linked immunosorbent assay (ELISA). Aquaculture, 42, 1–12.
576
577
6.
EMMENEGGER E.J., MEYERS T.R., BURTON T.O. & KURATH G. (2000). Genetic diversity and epidemiology of infectious hematopoietic necrosis virus in
Alaska. Dis. Aquat. Org., 40, 163–176.
578
579
7.
ENGELKING H.M., HARRY J.B. & LEONG J.C. (1991). Comparison of representative strains of infectious hematopoietic necrosis virus by serological
neutralization and cross-protection assays. Appl. Environ. Microbiol., 57, 1372–1378.
580
581
8.
ENZMANN P.J., KURATH G., FICHTNER D. & BERGMANN S.M. (2005). Infectious hematopoietic necrosis virus: Monophyletic origin of European IHNV
isolates from North-American genogroup M. Dis. Aquat. Org., 66, 187–195.
582
583
9.
GARVER K.A., BATTS W.N. & KURATH G. (2006). Virulence comparisons of infectious hematopoietic necrosis virus (IHNV) U and M genogroups in
sockeye salmon and rainbow trout. J. Aquat. Anim. Health, 18, 232–243.
584
585
10.
HUANG C., CHIEN M-S., LANDOLT M. & WINTON J.R. (1994). Characterization of the infectious hematopoietic necrosis virus glycoprotein using
neutralizing monoclonal antibodies. Dis. Aquat. Org., 18, 29–35.
586
587
588
11.
JORGENSEN P.E.V., OLESEN N.J., LORENZEN N., WINTON J.R. & RISTOW S.S. (1991). Infectious hematopoietic necrosis (IHN) and viral hemorrhagic
septicemia (VHS): detection of trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence, and
enzyme-linked immunosorbent assay. J. Aquat. Anim. Health, 3, 100–108.
589
590
12.
KIM W-S., OH M-J., NISHIZAWA T., PARK J-W., KURATH G. & YOSHIMIZU M. (2007). Genotyping of Korean isolates of infectious hematopoietic necrosis
virus (IHNV) based on the glycoprotein gene. Arch. Virol., 152, 2119–2124.
591
592
13.
KURATH G. (2005). Overview of Recent DNA Vaccine Development for Fish. In: Fish Vaccinology, Developments in Biologicals, Midtlyng P.J., ed.
Karger, Basel, Switzerland, 121, 201–213.
593
594
14.
KURATH G., GARVER K.A., TROYER R.M., EMMENEGGGER E.J., EINER-JENSEN K. & ANDERSON E.D. (2003). Phylogeography of infectious haematopoietic
necrosis virus in North America. J. Gen. Virol., 84, 803–814.
16
Manual of Diagnostic Tests for Aquatic Animals 2009
Chapter 2.1.4. – Infectious haematopoietic necrosis
595
596
15.
LAPATRA S.E., FRYER J.L. & ROHOVEC J.S. (1993). Virulence comparison of different electropherotypes of infectious hematopoietic necrosis virus.
597
598
16.
LAPATRA S.E., ROBERTI K.A., ROHOVEC J.S. & FRYER J.L. (1989). Fluorescent antibody test for the rapid diagnosis of infectious hematopoietic
necrosis virus. J. Aquat. Anim. Health, 1, 29–36.
599
600
17.
LAPATRA S.E., TURNER T., LAUDA K.A., JONES G.R. & WALKER S. (1993). Characterization of the humoral response of rainbow trout to infectious
hematopoietic necrosis virus. J. Aquat. Anim. Health, 5, 165–171.
601
602
18.
NISHIZAWA T., KINOSHITA S., KIM W-S., HIGASHI S. & YOSHIMIZU M. (2006). Nucleotide diversity of Japanese isolates of infectious hematopoietic
necrosis virus (IHNV) based on the glycoprotein gene. Dis. Aquat. Org., 71, 267-272.
603
604
19.
PURCELL M.K., HART S.A., KURATH G. & WINTON J.R. (2006). Strand-specific, real-time RT-PCR assays for quantification of genomic and positivesense RNAs of the fish rhabdovirus, infectious hematopoietic necrosis virus. J. Virol. Methods, 132, 18-24.
605
606
20.
RISTOW S.S. & ARNZEN J.M. (1989). Development of monoclonal antibodies that recognize a type 2 specific and a common epitope on the
nucleoprotein of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1, 119–125.
607
608
21.
RISTOW S.S. & ARNZEN DE AVILA J.M. (1991). Monoclonal antibodies to the glycoprotein and nucleoprotein of infectious hematopoietic necrosis
virus (IHNV) reveal differences among isolates of the virus by fluorescence, neutralization and electrophoresis. Dis. Aquat. Org., 11, 105–115.
609
610
22.
TROYER R.M. & KURATH G. (2003). Molecular epidemiology of infectious hematopoietic necrosis virus reveals complex virus traffic and evolution
within southern Idaho aquaculture. Dis. Aquat. Org., 55, 175–185.
611
612
23.
613
24.
WINTON J.R. (1997). Immunization with viral antigens: Infectious haematopoietic necrosis. Dev. Biol. Stand., 90, 211–220.
614
615
25.
WINTON J.R., ARAKAWA C.K., LANNAN C.N. & FRYER J.L. (1988). Neutralizing monoclonal antibodies recognize antigenic variants among isolates of
infectious hematopoietic necrosis virus. Dis. Aquat. Org., 4, 199–204.
616
617
26.
WINTON J.R. & EINER-JENSEN K. (2002). Molecular diagnosis of infectious hematopoietic necrosis and viral hemorrhagic septicemia. In: Molecular
Diagnosis of Salmonid Diseases, Cunningham C.O., ed. Kluwer, Dordrecht, The Netherlands, pp. 49–79.
618
619
27.
WOLF K. (1988). Infectious hematopoietic necrosis. In: Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, New York, USA, pp.
83–114
Dis. Aquat. Org., 16, 115–120.
WINTON J.R. (1991). Recent advances in the detection and control of infectious hematopoietic necrosis virus (IHNV) in aquaculture. Ann. Rev.
Fish Dis., 1, 83–93.
620
621
*
* *
622
623
NB: There is an OIE Reference Laboratory for infectious haematopoeitic necrosis (see Table at the end of this Aquatic Manual or consult the OIE
Web site for the most up-to-date list: www.oie.int).
Manual of Diagnostic Tests for Aquatic Animals 2009
17